PROJECT SUMMARY/ABSTRACT Cells must be organized with high fidelity in order to set up the body plan during development and minimize aberrant growth of cancer. By extension, when cells are lost to turnover or injury, patterning information in remaining tissue informs what cells need to be replaced. Some animals have a tremendous capacity to replace missing tissue through a process called regeneration. The regenerative flatworm, Schmidtea mediterranea, maintains its tissues through the proliferation of an adult stem cell population called neoblasts. During both homeostatic turnover and regeneration, neoblasts divide and give rise to the correct cell types according to their positon in regional gradients of gene expression in muscle. Predominant among these genes are components of the Wnt pathway. Disrupting these gradients of Wnt expression disrupts animal patterning, which manifests as misplaced and mis-proportioned tissue. The mechanistic link between the activity of regionally expressed gene products and local fate of both stem cells and differentiated cells is poorly understood. This study will examine how the activity of regionally expressed Wnts and Wnt regulators impacts cell fate in planarians, providing the first biochemical insights into how positional information is used in regeneration. Towards this goal, this proposal sets forth the following objectives: (1) To generate recombinant planarian Wnt pathway proteins and deliver them into animals by microinjection. The specific activities of these proteins will be assessed using in situ hybridizations studies with established regional and tissue markers. (2) To develop genomic editing techniques for planarians by delivery of Cas9 RNPs into neoblasts. Transgenic manipulations will allow for stable mis-expression of Wnts in non-native regions for the first time in regenerative animal. (3) To assess the effect of misplaced Wnt activity on neoblasts and differentiated tissue via a single cell sequencing approach. This analysis will show how different cell types respond to a changing Wnt environment and elucidate the functional relationships between patterning molecules and the cell fates they specify. Wnt signaling in planarians not only informs patterning during regeneration, it also contributes to maintenance of anatomical proportion in uninjured animals with tremendous fidelity. The mechanism of tissue scaling in planarians may also highlight potential checks on aberrant Wnt or somatic stem cell activity in other systems, such as cancer, where Wnt expression can be a predictor of tumor aggressiveness and metastases. Wnt signaling also underlies the self-renewal potential of the early mammalian heart and nervous system, two contexts of particular interest for regenerative medicine. Characterizing how cells respond to Wnt signaling to confer both developmental plasticity and disease states is therefore of broad biological and biomedical significance.